Pablo Moreno-Acosta

A non-radioactive PCR-SSCP analysis allows to distinguish between HPV 16 European and Asian-American variants in squamous cell carcinomas of the uterine cervix in Colombia

Human Papillomavirus type 16 (HPV 16) DNA is regularly found in around 50% of all cervical carcinomas. Variants of this type have been found associated with different risks for cervical cancer development. Presence of HPV 16 variants in Colombia has not been previously reported. The aims of this study were to assess the feasibility of non-radioactive PCR-SSCP (polymerase chain reaction single-strand conformation polymorphism) analysis for determination of variability of ORF of E6, variability in the enhancer sequence of the LCR, and for establishment of the distribution of HPV 16 variants in invasive squamous cell carcinoma of the uterine cervix in Colombian women. Biopsies from 59 patients at the Instituto Nacional de Cancerología (INC) in Bogotá (Colombia) were collected. HPV detection was performed using universal primers. HPV 16 variants were detected by non-radioactive single-stranded conformational polymorphism (SSCP) analysis and direct sequencing. HPV 16 was detected in 57.6% of the tumors. The European branch was identified in 88.2% of the samples with the E-G350 class being the most prevalent variant (41.1%). The Asian-American branch was identified in 8.8% of the samples. Within this group it was possible to distinguish between c and a classes. It was not possible to determine the branch in 2.9% of the cases. A nucleotide transition (G to A) at position 7521 was the most prevalent variation (80%) found in the enhancer sequence of the LCR region. Conclusion: A non-radioactive PCR-SSCP analysis allowed us to distinguish between European and Asian-American branches of HPV 16, and to distinguish among classes in squamous cell carcinomas of the uterine cervix in Colombia. This method is an excellent alternative that can be used as a screening tool for identification of HPV 16 variants.

Abstract C39: GLUT1 and hemoglobin levels: Hypoxic markers of treatment response in patients with locally advanced cervical cancer.

Background: The increased expression of GLUT1 and HKII CAIX in cervical cancer is associated with progression, metastasis, and poor survival rates. The aim of this study was to evaluate the predictive utility of GLUT1, CAIX, and HKII and hemoglobin levels versus tumor response to exclusive radiotherapy and concomitant chemoradiotherapy in patients with cervical carcinoma located. Methods: Sixty-six patients (FIGO IIB, IIIB) were included retrospectively for the period 2001-2007. Twenty-two of them received exclusive radiotherapy, and 44 received concurrent radiochemotherapy. The expression of GLUT1, CAIX, and HKII was studied by immunohistochemistry in paraffin-embedded biopsies taken before treatment. The expression levels of the proteins were correlated with clinical factors pathological, response to treatments (responders and non-responders), overall survival, and disease-free survival. Results: Of the 66 patients, 53 (80.3%) showed a complete response to treatment, of whom 16 received exclusive radiotherapy and 37 received concurrent radiochemotherapy. Protein expression levels of GLUT1, CAIX, and HKII were increased in cancer patients and as a particular feature of staining were observed that GLUT1 inmunostaining was frequently localized together to the blood vessels. Hemoglobin levels ≤ 11g/dl were statistically significant compared to the response to treatment (p=0.0127), with a trend of 4.3-fold risk of treatment failure in the group of nonresponders. We found that when expressed GLUT1, both the rate of overall survival and disease-free survival showed a trend risk of decrease of 1.1 times and 1.5 times respectively; when co-expressed GLUT1 and HKII was observed a trend risk of decrease of 1.6 times in the rate overall survival. Patients with hemoglobin levels ≤ 11g/dl had a risk 4.3-fold (p=0.02) decrease in both the overall survival rate as the rate of disease-free survival. Conclusion: The presence of anemic hypoxia (Hbg ≤ 11g/dl), expression of GLUT1, co-expression of GLUT1 and HKII, may influence the response to treatment, and therefore in overall and disease-free survival. The study and detection of these markers could contribute to infer the metabolic and hypoxic state of tumors, so that the therapeutic management may be optimized by considering such markers as predictive markers and/or as molecular targets. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C39. Citation Format: Pablo Moreno-Acosta, Alfredo Romero-Rojas, Schyrly Carrillo, Oscar Gamboa, Jinneth Acosta, Josep Balart-Serra, Nicolas Magne. GLUT1 and hemoglobin levels: Hypoxic markers of treatment response in patients with locally advanced cervical cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C39.

IGF-IR gene expression as a predictive marker of ionizing radiation response for patients with locally advanced cervical cancer

In Colombia the cancer of uterine cervix is diagnosed in advanced stages and its treatment is based on the application of the radiotherapy or radiotherapy more chemotherapy. Most of studies show that the IGF-IR expression leads to the cellular increase of resistance to radiation, that the tumor like hypoxia induces the IGF-IR expression and selects cells that overexpressing IGF-IR. The aim of this study was to evaluate the effect of expression of IGF-IR, IGF-I, IGF-II, GAPDH, and hemoglobin concentration on tumoral response to ionizing radiation in patients with locally advanced cervical cancer. A series of 37 consecutive patients were recruited from 2002 to 2004. For each patient, a representative tumoral sample in fresh was taken at the time of diagnosis before the beginning of the treatment. In order to have a group control for the analysis of gene expression, 30 samples of normal tissue of uterine cervix from patients treated by exclusive hysterectomy were also analyzed. The mean age of the remaining 37 patients was 46 years (ranging from 33 to 70 years). All patients received exclusive pelvic external beam radiotherapy (EBRT). Treatment consisted of teletherapy using 6 to 18 MV photons with standard four field technique delivering a total dose of 45— 50,4 Gy in 28 fractions (1.8 Gy per fraction, 5 consecutive days per week, overall EBRT treatment time 5 weeks). Follow-up was scheduled at 6 weeks after completion of intracavitary brachytherapy, then every three months during the next 2 years. Complete remission was defined as no evidence of residual disease on clinical examination and radiological imaging, six weeks to three months after completion of the therapeutic sequence. Gene expression of IGF-IR, IGF-I, IGF-II and GAPDH was determined by Real Time PCR and IGF-IR protein was detected using Western Blot. Hemoglobin levels were also evaluated as a parameter of oxygenation before the beginning of ionizing radiation (Hgb≤11g/dl). Gene expression levels were compared between complete responders and non responders using Anova or Kruskal-allis Anova. A major increase was observed in gene expression IGF-IR (34%), followed by the expression of IGF-II (24%) in the cases of cancer in comparison with the control group (expression not detected). Gene expression of IGF-IR (p=0.04) and incomplete treatment (p=0.019) were associated with the lack of treatment response. Patients expressing IGF-IR had 4.6 times more risk of non treatment response; GAPDH expression was directly correlated with the IGF-IR expression, IGF-IR and IGF-II co-expression under anemic hypoxia conditions (Hgb≤11g/dl), demonstrated a possible activation of a glicolytic pathway as an answer to the high metabolic rate of tumoral cells. This is the first report in the clinical setting that relates the expression of IGF-IR as a strong marker of responsiveness. Therefore, the IGF-IR can be considered as a molecular target for optimizing the treatment of cervical cancer with radiotherapy.

Abstract B85: IGF‐IR gene expression as a predictive marker of ionizing radiation response for patients with locally advanced cervical cancer

In Colombia the cancer of uterine cervix is diagnosed in advanced stages and its treatment is based on the application of the radiotherapy or radiotherapy more chemotherapy. Most of studies show that the IGF‐IR expression leads to the cellular increase of resistance to radiation, that the tumor like hypoxia induces the IGF‐IR expression and selects cells that overexpressing IGF‐IR. The aim of this study was to evaluate the effect of expression of IGF‐IR, IGF‐I, IGF‐II, GAPDH, and hemoglobin concentration on tumoral response to ionizing radiation in patients with locally advanced cervical cancer. A series of 37 consecutive patients were recruited from 2002 to 2004. For each patient, a representative tumoral sample in fresh was taken at the time of diagnosis before the beginning of the treatment. In order to have a group control for the analysis of gene expression, 30 samples of normal tissue of uterine cervix from patients treated by exclusive hysterectomy were also analyzed. The mean age of the remaining 37 patients was 46 years (ranging from 33 to 70 years). All patients received exclusive pelvic external beam radiotherapy (EBRT). Treatment consisted of teletherapy using 6 to 18 MV photons with standard four field technique delivering a total dose of 45–50,4 Gy in 28 fractions (1.8 Gy per fraction, 5 consecutive days per week, overall EBRT treatment time 5 weeks). Follow‐up was scheduled at 6 weeks after completion of intracavitary brachytherapy, then every three months during the next 2 years. Complete remission was defined as no evidence of residual disease on clinical examination and radiological imaging, six weeks to three months after completion of the therapeutic sequence. Gene expression of IGF‐IR, IGF‐I, IGF‐II and GAPDH was determined by Real Time PCR and IGF‐IR protein was detected using Western Blot. Hemoglobin levels were also evaluated as a parameter of oxygenation before the beginning of ionizing radiation (Hgb>10g/dl;Hgb Citation Information: Cancer Prev Res 2010;3(1 Suppl):B85.

Correction to: From IB2 to IIIB locally advanced cervical cancers: report of a ten-year experience

In the original publication [1] one author name was spelled incorrect.

Outcomes and treatments of IB1 cervical cancers with high recurrence risk: A 13 years’ experience

PURPOSE The aim of the present study was to identify management strategies and outcomes of patients with stage IB1 cervical cancer with high recurrence risk. MATERIALS AND METHODS Medical files of all consecutive patients treated between 2004 and 2017 with external beam radiotherapy and/or brachytherapy for IB1 cervical cancer, whatever the lymph node status, were retrospectively reviewed. RESULTS Forty-two patients were included, with a median age of 49.8 years old. Median tumour size, estimated with the initial pelvic magnetic resonance imaging, was 26mm (interquartile range [IQR]=19.5-35). Histological types were mainly squamous cell carcinoma (59.5%) and adenocarcinoma (33.3%). Lymphovascular invasion was reported for 38.1% of patients. Pelvic lymph nodes were involved for eight patients (19.0%). Surgery was performed for 39 patients (92.9%). A neoadjuvant treatment was delivered for 20 patients (47.6%), an adjuvant treatment for 19 patients (45.2%) and an exclusive radiotherapy (with or without chemotherapy) followed by brachytherapy for three patients (7.1%). Pathologic complete response was achieved in 61.5% of patients. With a median follow-up of 5.8 years (IQR=2.6-9.4), five patients (11.9%) experienced a tumour relapse. The five-year disease-free survival was 79.5% (95% confident interval [CI]=66.9-94.4), the five-year overall survival was 87.8% (95% CI=77.2-99.8), and the five-year disease-specific survival was 94.2% (95% CI=86.7-100). CONCLUSION In current clinical practice, tailored treatments are delivered, and seems to give correct therapeutic index. However, clinical trials are needed to standardise treatment according to patient characteristics and recurrence risk factors.

Abstract 435: Prognostic biomarkers as molecular targets for individualized neoadjuvant treatment for cervical cancer

Cervical cancer is one of the most prevalent malignancy and of higher mortality in the world, and is considered a marker of underdevelopment. Conventional radiotherapy is one of the treatments used for this type of cancer. 30 to 40% of patients with similar prognosis factors not respond equally to a comparable standard treatment. The poor response to radiotherapy leads to the development of innovative and effective therapies for cervical cancer locally advanced, metastatic and refractory. A comparative analysis of cervical cancer in the context of other cancers may reveal that it is relatively smaller number of targeted molecular agents that have been tested. Accordingly, a number of biological agents are currently in clinical development for the purpose of, inhibiting angiogenesis, molecularly address EGFR and IGF-1R, modulation of cell cycle, of histone deacetylases, COX-2, mTOR and tumor microenvironment (hypoxia and glycolysis). Within work that we have been developing, reported that gene expression of IGF1R is a strong predictive marker for lack of response to radiotherapy, patients have 28.6 times higher risk of failure treatment; Objective: To determine whether expression of IGF-IR, GAPDH, HIF-1 alpha, Survivin, GLUT1, CAIX, HKII and clinicopathological parameters can be used as prognostic biomarkers to treatment outcome and as possible molecular targets. Patients & Methods: This prospective cohort study included 149 patients with squamous cell carcinomas of the uterine cervix in FIGO stages IIB and IIIB between 2008 and 2011. The mean age was 46 years. Of the 149 patients, 61 were treated with radiotherapy and 88 with concurrent radiochemotherapy. Expression of the proteins CAIX, GLUT-1, HIF1α, HKII, IGF-IRα, IGF-IRβ and Survivin was determined by immunohistochemistry in biopsies taken before treatment. Results: The highest increase was found in expression of GAPDH (100%), Survivin (87%), followed of, IGF-IRα (76.5%), IGF-IRβ (74.5%), IGF-IRα and IGF-IRβ concordance in the expression(73%), HIF1α (74.1%); strong expression was observed with low frequency for GLUT-1 (31.1%), CAIX (16.2%), HKII (10.6%). We found that patients who do not express IGF-1Rs, GLUT1 and having hemoglobin levels > 11g/dl have improved overall survival compared to those that express IGF-1Rs, GLUT1 and having hemoglobin ≤ 11g/dl (P = 0.0158). Conclusions: Using the expression of GLUT1, IGF-1Rs and Hb levels (≤ 11g/dl) as therapeutic molecular targets could contribute to an appropriate therapeutic management as individualized neoadjuvant treatment for cervical cancer Citation Format: Pablo Moreno-Acosta, Oscar Gamboa, Diana Mayorga, Alfredo Romero, Jinneth Acosta, Maria Carolina Sanabria, Martha Cotes Mestre, Nicolas Magne. Prognostic biomarkers as molecular targets for individualized neoadjuvant treatment for cervical cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 435.

Abstract B10: Predictive and prognostic biomarkers at personalized treatment of locally advanced cervical cancer

Introduction: Cervical cancer is one of the most prevalent malignancy and of higher mortality in the world, and is considered a marker of underdevelopment. Conventional radiotherapy is one of the treatments used for this type of cancer. 30 to 40% of patients with similar prognosis factors not respond equally to a comparable standard treatment. The poor response to radiotherapy leads to the development of innovative and effective therapies for cervical cancer locally advanced, metastatic and refractory. A comparative analysis of cervical cancer in the context of other cancers may reveal that it is relatively smaller number of targeted molecular agents that have been tested. Accordingly, a number of biological agents are currently in clinical development for the purpose of, inhibiting angiogenesis, molecularly address EGFR and IGF-1R, modulation of cell cycle, of histone deacetylases, COX-2, mTOR and tumor microenvironment (hypoxia and glycolysis). Within work that we have been developing, reported that gene expression of IGF1R is a strong predictive marker for lack of response to radiotherapy, patients have 28.6 times higher risk of failure treatment; Patients & Methods: This prospective cohort study included 149 patients with squamous cell carcinomas of the uterine cervix in FIGO stages IIB and IIIB between 2008 and 2011. The mean age was 46 years. Of the 149 patients, 61 were treated with radiotherapy and 88 with concurrent radiochemotherapy. Expression of the proteins CAIX, GLUT-1, HIF1α, HKII, IGF-IRα, IGF-IRβ and Survivin was determined by immunohistochemistry and P53 Arg72Pro polymorphism by allele specific PCR in biopsies taken before treatment. Results: The highest increase was found in expression of GAPDH (100%), Survivin (87%), followed of, IGF-IRα (76.5%), IGF-IRβ (74.5%), IGF-IRα and IGF-IRβ concordance in the expression(73%), HIF1α (74.1%); strong expression was observed with low frequency for GLUT-1 (31.1%), CAIX (16.2%), HKII (10.6%). For polymorphism Arg72Pro of P53 was found that genotype Arg/Arg is the most frequently (59.8) in patients with cervical locally advanced cancer and that compared to the control group (non-tumor tissue of the cervix with histopathologic diagnosis of cervicitis without evidence of squamous intraepithelial lesion or malignancy) (P = 0.000), and published reports, his presence confirms their possible participation in the development of this cancer, however we found no association with the outcome and response to treatment. Patients who received chemoradiotherapy compared to those who received radiotherapy showed higher survival times, both for overall survival and disease-free. Patients with hemoglobin levels ≤ 11g/dl had a risk 2.53-fold (p = 0.01) decrease in both the overall survival rate as the rate of disease-free survival. Conclusions: We found that the expression of IGF-IRβ detected by immunohistochemistry is a prognostic marker that affects overall survival and disease-free survival; the detection and study before treatment of the expression of HIF1α, CAIX, GLUT 1 and HKII, considered as biological factors pre-existing, contributes to infer the metabolic and hypoxic state, as also at the rational use of new modalities in radiotherapy, radiochemotherapy and gene therapy in the regulation of hypoxia. Citation Format: Pablo Moreno-Acosta, Oscar Gamboa, Alfredo Romero-Rojas, Jinneth Acosta, Martha Cotes Mestre, Diana Mayorga, Magda Pacheco, Gladys Aguilera, Nicolas Magne. Predictive and prognostic biomarkers at personalized treatment of locally advanced cervical cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B10.

Abstract C88: HPV infections, hTERT methylation and hTERT expression in patients with invasive cervical cancer

Background: There is limited information of the role of hTERT methylation, hTERT expression and their association with type specific HPV infections in patients with invasive cervical cancer Objective: To analyze the possible association between methylation status of the hTERT promoter gene, hTERT expression and HPV infection in patients with invasive cervical cancer. Methodology: Eighty seven frozen samples of women with invasive cervical cancer were analyzed for type specific HPV infection using a GP5+/GP6+ mediated PCR- RLB. hTERT DNA methylation analysis was performed on bisulfite modified DNA using a new PCR-RLB-hTERT methylation assay targeting two regions flanking the hTERT [region 1 (nt -240 to -1) and region 2 (nt +1 to +120) relative to first ATG]. Expression of hTERT was detected by immunohistochemistry using an Anti-hTERT (348-358) rabbit pAb). Results: All samples with HPV types belonging to the α7 species (HPV 18, 45 and 59) showed no hTERT methylation in region 1 (core promoter) and an increase in% of partial methylation in region 2. Samples with HPV types belonging to the α9 specie (16, 52, 35, 31 and 58) showed similar% of methylation in region 1 and region 2 according viral type but the percentages of methylation were different between them (ranging from 0% for HPV58 samples to 66.7% for HPV31). Strong expression of hTERT was observed in 77% of the samples independent of the HPV genotype. Conclusion: hTERT methylation seems to be associated with specific genotype HPV infection; however expression of the hTERT protein seems to depend of additional molecular events and worth further investigation. Citation Format: Pablo Moreno-Acosta, Nicolas Morales, Marcela Burgos, Oscar Gamboa, Juan Carlos Mejia, Alfredo Romero-Rojas, Alba Lucia Combita, Monica Molano. HPV infections, hTERT methylation and hTERT expression in patients with invasive cervical cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C88.

369 Diagnosis and molecular targeting for individualized treatment of patients with pre-neoplastic lesions and locally advanced cervical cancer

Background: The natural history of HPV infection and development of cervical intraepithelial neoplasia indicate that most lesions disappear without treatment in contrast to a significant proportion of high-grade lesions that progress to invasive cancer if not treated. Persistent infection with high-risk HPV may be necessary for the development of cervical cancer. The purpose of the study was make a retrospective molecular diagnosis that include detection of HPV16 variants, polymorphism Arg72Pro of P53, expression of anti-apoptotic markers such as IGF-1R and Survivin, and hypoxic markers and glycolytic as GLUT1 and CAIX and markers of progression as hTERT in samples invasive cancer and preneoplastic lesions taken in 2002 and 1986; the results may be useful to propose an appropriate therapeutic management.